Project

NMR; Coordination Chemistry; Metal Ions; Mammalian Ribozyme; Splicing; Group II Intron Ribozyme

Lead
Lay summary
Metal ions are necessary for folding and function of catalytic RNA molecules. However, the structural and mechanistic roles of these ions are still largely in the dark. Na(I) and Mg(II) are usually assumed to be the sole metallic cofactors involved with nucleic acids in living systems, both being most abundant and freely available in the cell. The recognition of these and other metal ions by nucleic acids is poorly understood as is their effect on structure, folding, and catalytic activity. This study focuses on the metal ion assisted assembly of the catalytic core of two different ribozymes, i.e. a group II intron from yeast as well as a mammalian ribozyme, and the thermodynamic and structural characterization of these interactions. A multidisciplinary approach is applied by a combination of tools from Coordination and Analytical Chemistry as well as Structural Biology, mainly biomolecular NMR spectroscopy. Our results will not only contribute to the understanding of the metal ion-promoted structure, folding, and mechanism of the RNAs catalytic core, but also promise to have a significant impact on the Biological Inorganic Chemistry of RNAs and on RNA Biochemistry in general.

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Last update: 21.02.2013

Active participation

Self-organised

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1.SUMMARY AND CENTRAL AIMS OF THE STUDYMetal ions are necessary for folding and function of catalytic RNA molecules. However, the structural and mechanistic roles of these ions are still largely in the dark. Na+ and Mg2+ are usually assumed to be the sole metallic cofactors involved with nucleic acids in living systems, both being most abundant and freely available in the cell. The recognition of these and other metal ions by nucleic acids is poorly understood as is their effect on structure, folding, and catalytic activity. This study focuses on the metal ion assisted assembly of the catalytic core of two different ribozymes, i.e., group II introns and the mammalian CPEB3 ribozyme, and the thermodynamic and structural characterization of these interactions. A multidisciplinary approach is applied by a combination of tools from Coordination and Analytical Chemistry as well as Structural Biology. Our results will not only contribute to the understanding of the metal ion-promoted structure, folding, and mechanism of the RNAs catalytic core, but also promise to have a significant impact on the Biological Inorganic Chemistry of RNAs and on RNA Biochemistry in general. AIM A: Understanding the Assembly of the Group II Intron Catalytic CoreGroup II introns are among the largest occurring RNAs in Nature and are closely related to the eukaryotic spliceosomal machinery. These self-splicing introns exhibit a straight folding pathway devoid of kinetic traps. Folding is initiated by Mg2+ coordination, whereby the kappa-zeta region within domain 1 (D1) is the key element for the first phase of folding. The last step of folding is the Mg2+-assisted docking of domain 5 (D5). We have just solved the solution structure of this central kappa-zeta region and characterized its structural change upon Mg2+ binding. It turned out that an additional extended helix of kappa-zeta is needed for D5 docking: Here we concentrate on the folding, structure and rearrangement of this extended kappa-zeta region. We will first investigate the Mg2+ requirement and structural changes within this three-way junction. Second, the Mg2+-dependent association of the extended kappa-zeta RNA with D5, the specific Mg2+ coordination, as well as the structure of the about 100 nucleotide long extended kappa-zeta/D5 complex will be characterized and solved by NMR. This RNA is unique and perfectly suited for bioinorganic studies because of its high affinity for Mg2+ and the associated structural change. In addition, it also represents a minimal catalytic core of group II introns.AIM B: Folding, Structure, and Mechanism of the Mammalian CPEB3 RibozymeThe CPEB3 ribozyme is an intronic sequence within the cytoplasmatic polyadenylation element-binding protein 3. This RNA belongs to the class of hepatitis delta virus (HDV)-like self-cleaving ribozymes and is special because it is one of two ribozymes occurring in mammals. The human homologue is polymorphic in sequence whereby the nature of one specific nucleotide is linked to cleavage activity and the performance of episodic memory tasks. Like all RNAs, also structure and function of HDV-like ribozymes are strictly dependent on metal ions. Here we will concentrate on the elucidation and characterization of the interplay between different mono- and divalent metal ions and the polymorphs of the ribozyme with regard to folding, structure and mechanism. X-ray crystallography and NMR will be mainly applied to elucidate the structure, determine the nature of metal ion-binding pockets, as well as their role in folding and catalytic core formation. Our ultimate goal is to understand the coordination-chemical basis of the accelerating or inhibiting influence of metal ions in a motion-structure-function relationship of this unique mammalian ribozyme.AIM C: Metal Ion Coordination at Atomic ResolutionTo elucidate the exact coordination pattern of metal ions is a highly challenging but central goal of our research: Based on the solved NMR structures under AIMs A and B, we apply further methods to understand metal ion binding in detail and to correlate it directly to structural changes and possibly catalysis of the RNAs. In combination with the Metal Ions in Nucleic Acids (MINAS) database, this will allow us to establish general rules for the roles of metal ions in nucleic acids.